U.S. patent application number 13/725772 was filed with the patent office on 2013-07-11 for accelerated wright-giemsa and may-grunwald staining methods.
This patent application is currently assigned to ABBOTT LABORATORIES. The applicant listed for this patent is ABBOTT LABORATORIES. Invention is credited to Rene Nieves Alicea, Koshy T. Chacko, Abe S. Mamaghani, Rupa Rao.
Application Number | 20130177974 13/725772 |
Document ID | / |
Family ID | 48698794 |
Filed Date | 2013-07-11 |
United States Patent
Application |
20130177974 |
Kind Code |
A1 |
Mamaghani; Abe S. ; et
al. |
July 11, 2013 |
Accelerated Wright-Giemsa and May-Grunwald Staining Methods
Abstract
The present disclosure provides methods for carrying out
Romanowsky-type stains, specifically Wright-Giemsa and May-Gr nwald
stains, quickly and efficiently. The methods greatly reduce the
overall amount of time required to complete a Wright-Giemsa stain
or a May-Gr nwald stain of sufficient quality on a biological
sample. The subject methods can be applied to both manual and
automated staining procedures.
Inventors: |
Mamaghani; Abe S.; (San
Jose, CA) ; Chacko; Koshy T.; (San Jose, CA) ;
Rao; Rupa; (Los Gatos, CA) ; Alicea; Rene Nieves;
(San Francisco, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ABBOTT LABORATORIES; |
Abbott Park |
CA |
US |
|
|
Assignee: |
ABBOTT LABORATORIES
Abbott Park
CA
|
Family ID: |
48698794 |
Appl. No.: |
13/725772 |
Filed: |
December 21, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61581040 |
Dec 28, 2011 |
|
|
|
61581042 |
Dec 28, 2011 |
|
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Current U.S.
Class: |
435/288.7 |
Current CPC
Class: |
G01N 1/30 20130101; G01N
2001/302 20130101; Y10T 436/2575 20150115; G01N 1/312 20130101;
G01N 2001/307 20130101 |
Class at
Publication: |
435/288.7 |
International
Class: |
G01N 1/31 20060101
G01N001/31 |
Claims
1. An automated staining system for performing a Wright-Giemsa
stain on one or more samples, comprising: a sample application
subsystem that applies a sample to a substrate; a fixation reagent
bath; a Wright-Giemsa staining reagent bath; a rinse reagent bath;
a sample transfer subsystem for moving the substrate from the
sample application subsystem, to the fixation reagent bath, to the
Wright-Giemsa staining bath, and then to the rinse reagent bath;
and a computer readable storage medium comprising instructions
executable by at least one processing device that, when executed,
cause the processing device to control the sample transfer
subsystem such that the sample transfer subsystem places the one or
more samples in the fixation reagent bath for a period of time
ranging from about 15 seconds up to about 45 seconds, in the
Wright-Giemsa staining reagent bath for a period of time ranging
from about 15 seconds up to about 60 seconds, and in the rinse
reagent bath for a period of time ranging from about 105 seconds up
to about 135 seconds.
2. The system of claim 1, wherein the sample is placed in the
fixation reagent bath for a period of time ranging from about 20
seconds up to about 40 seconds.
3. The system of claim 1, wherein the sample is placed in the
fixation reagent bath for a period of time ranging from about 25
seconds up to about 35 seconds.
4. The system of claim 1, wherein the fixation reagent is
methanol.
5. The system of claim 1, wherein the sample is placed in the
Wright-Giemsa staining reagent bath for a period of time ranging
from about 20 seconds up to about 50 seconds.
6. The system of claim 1, wherein the sample is placed in the
Wright-Giemsa staining reagent bath for a period of time ranging
from about 25 seconds up to about 45 seconds.
7. The system of claim 1, wherein the Wright-Giemsa staining
reagent comprises methanol.
8. The system of claim 1, wherein the sample is placed in the rinse
reagent bath for a period of time ranging from about 110 seconds up
to about 130 seconds.
9. The system of claim 1, wherein the sample is placed in the rinse
reagent bath for a period of time ranging from about 115 seconds up
to about 125 seconds.
10. The system of claim 1, wherein the rinse reagent comprises
de-ionized water.
11. The system of claim 1, wherein the rinse reagent is a phosphate
buffer.
12. The system of claim 11, wherein the phosphate buffer is a high
salt phosphate buffer.
13. The system of claim 11, wherein the phosphate buffer is a low
salt phosphate buffer.
14. The system of claim 11, wherein the phosphate buffer has a pH
value ranging from about 5.0 up to about 9.0 pH units.
15. The system of claim 11, wherein the phosphate buffer has a pH
value ranging from about 6.5 up to about 7.0 pH units.
16. The system of claim 1, further comprising placing the sample in
a second rinse reagent bath for a period of time ranging from about
5 seconds up to about 30 seconds.
17. The system of claim 16, wherein the sample is placed in the
second rinse reagent bath for a period of time ranging from about
10 seconds up to about 25 seconds.
18. The system of claim 16, wherein the sample is placed in the
second rinse reagent bath for a period of time ranging from about
10 seconds up to about 20 seconds.
19. The system of claim 16, wherein the second rinse reagent
comprises de-ionized water.
20. The system of claim 16, wherein the second rinse reagent is a
phosphate buffer.
21. The system of claim 20, wherein the phosphate buffer is a high
salt phosphate buffer.
22. The system of claim 20, wherein the phosphate buffer is a low
salt phosphate buffer.
23. The system of claim 20, wherein the phosphate buffer has a pH
value ranging from about 5.0 up to about 9.0 pH units.
24. The system of claim 20, wherein the phosphate buffer has a pH
value ranging from about 6.5 up to about 7.0 pH units.
25. The system of claim 1, further comprising placing the sample in
a drying chamber.
26. The system of claim 1, wherein the substrate is a glass
microscope slide.
27. The system of claim 1, wherein the sample is a biological
fluid.
28. The system of claim 1, wherein the sample is a blood
sample.
29. The system of claim 1, wherein the sample is a bone marrow
sample.
30. An automated staining system for performing a May-Gr nwald
stain on one or more samples, comprising: a sample application
subsystem that applies a sample to a substrate; a fixation reagent
bath; a May-Gr nwald staining reagent bath; a rinse reagent bath; a
sample transfer subsystem for moving the substrate from the sample
application subsystem, to the fixation reagent bath, to the May-Gr
nwald staining bath, and then to the rinse reagent bath; and a
computer readable storage medium comprising instructions executable
by at least one processing device that, when executed, cause the
processing device to control the sample transfer subsystem such
that the sample transfer subsystem places the one or more samples
in the fixation reagent bath for a period of time ranging from
about 15 seconds up to about 45 seconds, in the May-Gr nwald
staining reagent bath for a period of time ranging from about 165
seconds up to about 195 seconds, and in the rinse reagent bath for
a period of time ranging from about 135 seconds up to about 165
seconds.
31. The system of claim 30, wherein the sample is placed in the
fixation reagent bath for a period of time ranging from about 20
seconds up to about 40 seconds.
32. The system of claim 30, wherein the sample is placed in the
fixation reagent bath for a period of time ranging from about 25
seconds up to about 35 seconds.
33. The system of claim 30, wherein the fixation reagent is
methanol.
34. The system of claim 30, wherein the sample is placed in the
May-Gr nwald staining reagent bath for a period of time ranging
from about 170 seconds up to about 190 seconds.
35. The system of claim 30, wherein the sample is placed in the
May-Gr nwald staining reagent bath for a period of time ranging
from about 175 seconds up to about 185 seconds.
36. The system of claim 30, wherein the May-Gr nwald staining
reagent comprises methanol.
37. The system of claim 30, wherein the sample is placed in the
rinse reagent bath for a period of time ranging from about 140
seconds up to about 160 seconds.
38. The system of claim 30, wherein the sample is placed in the
rinse reagent bath for a period of time ranging from about 145
seconds up to about 155 seconds.
39. The system of claim 30, wherein the rinse reagent comprises
de-ionized water.
40. The system of claim 30, wherein the rinse reagent is a
phosphate buffer.
41. The system of claim 40, wherein the phosphate buffer is a high
salt phosphate buffer.
42. The system of claim 40, wherein the phosphate buffer is a low
salt phosphate buffer.
43. The system of claim 40, wherein the phosphate buffer has a pH
value ranging from about 5.0 up to about 9.0 pH units.
44. The system of claim 40, wherein the phosphate buffer has a pH
value ranging from about 6.5 up to about 7.0 pH units.
45. The system of claim 30, further comprising placing the sample
in a second rinse reagent bath for a period of time ranging from
about 15 seconds up to about 45 seconds.
46. The system of claim 45, wherein the sample is placed in the
second rinse reagent bath for a period of time ranging from about
20 seconds up to about 40 seconds.
47. The system of claim 45, wherein the sample is placed in the
second rinse reagent bath for a period of time ranging from about
25 seconds up to about 35 seconds.
48. The system of claim 45, wherein the second rinse reagent
comprises de-ionized water.
49. The system of claim 45, wherein the second rinse reagent is a
phosphate buffer.
50. The system of claim 49, wherein the phosphate buffer is a high
salt phosphate buffer.
51. The system of claim 49, wherein the phosphate buffer is a low
salt phosphate buffer.
52. The system of claim 49, wherein the phosphate buffer has a pH
value ranging from about 5.0 up to about 9.0 pH units.
53. The system of claim 49, wherein the phosphate buffer has a pH
value ranging from about 6.5 up to about 7.0 pH units.
54. The system of claim 30, further comprising placing the sample
in a drying chamber.
55. The system of claim 30, wherein the substrate is a glass
microscope slide.
56. The system of claim 30, wherein the sample is a biological
fluid.
57. The system of claim 30, wherein the sample is a blood
sample.
58. The system of claim 30, wherein the sample is a bone marrow
sample.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] Under 35 U.S.C. .sctn.119(e), this application claims
priority to the filing date of U.S. Provisional Patent Application
Ser. No. 61/581,040, filed on Dec. 28, 2011, the disclosure of
which application is herein incorporated by reference in its
entirety, and to U.S. Provisional Patent Application Ser. No.
61/581,042, filed on Dec. 28, 2011, the disclosure of which
application is herein incorporated by reference in its
entirety.
BACKGROUND
[0002] In the field of tissue staining, there is a type of stain
generally referred to as a Romanowsky stain. Romanowsky is
generally credited with the discovery of the unique staining
effects of thiazin and eosin dyes on blood in 1891. D. Romanowsky
(1891) Zur Frage der Parisitologie und Therapie der Malaria, St
Petersb. Med. Wschr. 16, 297-302, and D. Romanowsky (1891) Zur
Frage der Parisitologie und Therapie der Malaria, St Petersb. Med.
Wschr 16, 307-15. Since that time, many variations of Romanowsky
staining have been proposed and created. See, e.g., P. N. Marshall,
Romanowsky-Type Stains in Hematology, Histochem. J. 10, 1-29
(1978). Modern methods also include those developed by James Wright
in the U.S. and Gustav Giemsa in Europe. Combination stains, known
as the Wright-Giemsa and May-Gr nwald-Giemsa (Pappenheim) stains,
are also now widely used, and generally employ mixtures of thiazin
and eosin dyes.
[0003] The present disclosure provides methods for carrying out
Romanowsky-type stains, specifically Wright-Giemsa stains and
May-Gr nwald stains, quickly and efficiently. The methods provided
herein greatly reduce the overall amount of time required to
complete a Wright-Giemsa stain or a May-Gr nwald stain of
sufficient quality on a biological sample. The subject methods can
be applied to manual as well as automated staining procedures.
SUMMARY
[0004] The present disclosure provides methods for carrying out
Romanowsky-type stains, specifically Wright-Giemsa stains and
May-Gr nwald stains, quickly and efficiently. The methods provided
herein greatly reduce the overall amount of time required to
complete a Wright-Giemsa stain or a May-Gr nwald stain of
sufficient quality on a biological sample. The subject methods can
be applied to manual as well as automated staining procedures.
[0005] In some embodiments, the present disclosure provides an
automated staining system for performing a Wright-Giemsa stain on
one or more samples, the system including a sample application
subsystem that applies a sample to a substrate; a fixation reagent
bath; a Wright-Giemsa staining reagent bath; a rinse reagent bath;
a sample transfer subsystem for moving the substrate from the
sample application subsystem, to the fixation reagent bath, to the
Wright-Giemsa staining bath, and then to the rinse reagent bath;
and a computer readable storage medium comprising instructions
executable by at least one processing device that, when executed,
cause the processing device to control the sample transfer
subsystem such that the sample transfer subsystem places the one or
more samples in the fixation reagent bath for a period of time
ranging from about 15 seconds up to about 45 seconds, in the
Wright-Giemsa staining reagent bath for a period of time ranging
from about 15 seconds up to about 60 seconds, and in the rinse
reagent bath for a period of time ranging from about 105 seconds up
to about 135 seconds.
[0006] In some embodiments, the sample is placed in the fixation
reagent bath for a period of time ranging from about 20 seconds up
to about 40 seconds. In some embodiments, the sample is placed in
the fixation reagent bath for a period of time ranging from about
25 seconds up to about 35 seconds. In some embodiments, the
fixation reagent is methanol.
[0007] In some embodiments, the sample is placed in the
Wright-Giemsa staining reagent bath for a period of time ranging
from about 20 seconds up to about 50 seconds. In some embodiments,
the sample is placed in the Wright-Giemsa staining reagent bath for
a period of time ranging from about 25 seconds up to about 45
seconds. In some embodiments, the Wright-Giemsa staining reagent
comprises methanol.
[0008] In some embodiments, the sample is placed in the rinse
reagent bath for a period of time ranging from about 110 seconds up
to about 130 seconds. In some embodiments, the sample is placed in
the rinse reagent bath for a period of time ranging from about 115
seconds up to about 125 seconds. In some embodiments, the rinse
reagent comprises de-ionized water. In some embodiments, the rinse
reagent is a phosphate buffer. In some embodiments, the phosphate
buffer is a high salt phosphate buffer. In some embodiments, the
phosphate buffer is a low salt phosphate buffer. In some
embodiments, the phosphate buffer has a pH value ranging from about
5.0 up to about 9.0 pH units. In some embodiments, the phosphate
buffer has a pH value ranging from about 6.5 up to about 7.0 pH
units.
[0009] In some embodiments, the sample is placed in a second rinse
reagent bath for a period of time ranging from about 5 seconds up
to about 30 seconds. In some embodiments, the sample is placed in
the second rinse reagent bath for a period of time ranging from
about 10 seconds up to about 25 seconds. In some embodiments, the
sample is placed in the second rinse reagent bath for a period of
time ranging from about 10 seconds up to about 20 seconds. In some
embodiments, the second rinse reagent comprises de-ionized water.
In some embodiments, the second rinse reagent is a phosphate
buffer. In some embodiments, the phosphate buffer is a high salt
phosphate buffer. In some embodiments, the phosphate buffer is a
low salt phosphate buffer. In some embodiments, the phosphate
buffer has a pH value ranging from about 5.0 up to about 9.0 pH
units. In some embodiments, the phosphate buffer has a pH value
ranging from about 6.5 up to about 7.0 pH units. In some
embodiments, the sample is placed in a drying chamber.
[0010] In some embodiments, the substrate is a glass microscope
slide. In some embodiments, the sample is a biological fluid. In
some embodiments, the sample is a blood sample. In some
embodiments, the sample is a bone marrow sample.
[0011] In some embodiments, the present disclosure provides an
automated staining system for performing a May-Gr nwald stain on
one or more samples, the system including a sample application
subsystem that applies a sample to a substrate; a fixation reagent
bath; a May-Gr nwald staining reagent bath; a rinse reagent bath; a
sample transfer subsystem for moving the substrate from the sample
application subsystem, to the fixation reagent bath, to the May-Gr
nwald staining bath, and then to the rinse reagent bath; and a
computer readable storage medium comprising instructions executable
by at least one processing device that, when executed, cause the
processing device to control the sample transfer subsystem such
that the sample transfer subsystem places the one or more samples
in the fixation reagent bath for a period of time ranging from
about 15 seconds up to about 45 seconds, in the May-Gr nwald
staining reagent bath for a period of time ranging from about 165
seconds up to about 195 seconds, and in the rinse reagent bath for
a period of time ranging from about 135 seconds up to about 165
seconds.
[0012] In some embodiments, the sample is placed in the fixation
reagent bath for a period of time ranging from about 20 seconds up
to about 40 seconds. In some embodiments, the sample is placed in
the fixation reagent bath for a period of time ranging from about
25 seconds up to about 35 seconds. In some embodiments, the
fixation reagent is methanol.
[0013] In some embodiments, the sample is placed in the May-Gr
nwald staining reagent bath for a period of time ranging from about
170 seconds up to about 190 seconds. In some embodiments, the
sample is placed in the May-Gr nwald staining reagent bath for a
period of time ranging from about 175 seconds up to about 185
seconds. In some embodiments, the May-Gr nwald staining reagent
comprises methanol.
[0014] In some embodiments, the sample is placed in the rinse
reagent bath for a period of time ranging from about 140 seconds up
to about 160 seconds. In some embodiments, the sample is placed in
the rinse reagent bath for a period of time ranging from about 145
seconds up to about 155 seconds. In some embodiments, the rinse
reagent comprises de-ionized water. In some embodiments, the rinse
reagent is a phosphate buffer. In some embodiments, the phosphate
buffer is a high salt phosphate buffer. In some embodiments, the
phosphate buffer is a low salt phosphate buffer. In some
embodiments, the phosphate buffer has a pH value ranging from about
5.0 up to about 9.0 pH units. In some embodiments, the phosphate
buffer has a pH value ranging from about 6.5 up to about 7.0 pH
units.
[0015] In some embodiments, the sample is placed in a second rinse
reagent bath for a period of time ranging from about 15 seconds up
to about 45 seconds. In some embodiments, the sample is placed in
the second rinse reagent bath for a period of time ranging from
about 20 seconds up to about 40 seconds. In some embodiments, the
sample is placed in the second rinse reagent bath for a period of
time ranging from about 25 seconds up to about 35 seconds. In some
embodiments, the second rinse reagent comprises de-ionized water.
In some embodiments, the second rinse reagent is a phosphate
buffer. In some embodiments, the phosphate buffer is a high salt
phosphate buffer. In some embodiments, the phosphate buffer is a
low salt phosphate buffer. In some embodiments, the phosphate
buffer has a pH value ranging from about 5.0 up to about 9.0 pH
units. In some embodiments, the phosphate buffer has a pH value
ranging from about 6.5 up to about 7.0 pH units.
[0016] In some embodiments, the substrate is a glass microscope
slide. In some embodiments, the sample is a biological fluid. In
some embodiments, the sample is a blood sample. In some
embodiments, the sample is a bone marrow sample.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1 is an illustration of a cartridge that can be used to
hold several substrates at the same time and move the substrates
between portions of a staining system or subsystem.
[0018] FIG. 2 is an illustration of a portion of a staining system
showing several reagent bath containers and several cartridges
holding multiple substrates.
[0019] FIG. 3 is illustration of a staining system showing movement
of a cartridge holding multiple substrates through several reagent
baths, followed by movement to a drying area.
[0020] FIG. 4: Panel A is a microscopic image of a sample that was
stained using a high salt phosphate buffer. Panel B is a
microscopic image of a sample that was stained using a low salt
phosphate buffer.
[0021] FIG. 5 is a graph showing stain quality as a function of the
pH of the rinse reagent used in a May-Gr nwald staining method of
the present disclosure.
[0022] FIG. 6 is a graph showing stain quality as a function of the
pH of the rinse reagent used in a Wright-Giemsa staining method of
the present disclosure.
[0023] FIG. 7: Panel A is a graph showing the reproducibility of a
Wright-Giemsa staining method that was performed over a period of
days. Panel B is a graph showing the reproducibility of a May-Gr
nwald staining method that was performed over a period of days.
[0024] FIG. 8: Panel A is a graph showing the numerical score of
cell by cell rating for a number of samples that were stained using
a Wright-Giemsa staining method. Panel B is a graph showing the
numerical score of cell by cell rating for a number of samples that
were stained using a May-Gr nwald staining method.
DETAILED DESCRIPTION
[0025] The present disclosure provides methods for carrying out
Romanowsky-type stains, specifically Wright-Giemsa stains and
May-Gr nwald stains, quickly and efficiently. The methods provided
herein greatly reduce the overall amount of time required to
complete a Wright-Giemsa stain or a May-Gr nwald stain of
sufficient quality on a biological sample. The subject methods can
be applied to manual as well as automated staining procedures.
DEFINITIONS
[0026] Before the present invention is further described, it is to
be understood that this invention is not limited to particular
embodiments described, as such may, of course, vary. It is also to
be understood that the terminology used herein is for the purpose
of describing particular embodiments only, and is not intended to
be limiting, since the scope of the present invention will be
limited only by the appended claims.
[0027] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limit of that range and any other stated or intervening
value in that stated range, is encompassed within the invention.
The upper and lower limits of these smaller ranges may
independently be included in the smaller ranges, and are also
encompassed within the invention, subject to any specifically
excluded limit in the stated range. Where the stated range includes
one or both of the limits, ranges excluding either or both of those
included limits are also included in the invention.
[0028] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can also be used in the practice or testing of the present
invention, the preferred methods and materials are now described.
All publications mentioned herein are incorporated herein by
reference to disclose and describe the methods and/or materials in
connection with which the publications are cited.
[0029] It must be noted that as used herein and in the appended
claims, the singular forms "a," "an," and "the" include plural
referents unless the context clearly dictates otherwise. Thus, for
example, reference to "a sample" includes a plurality of such
samples and reference to "the sample" includes reference to one or
more samples and equivalents thereof known to those skilled in the
art, and so forth. It is further noted that the claims may be
drafted to exclude any optional element. As such, this statement is
intended to serve as antecedent basis for use of such exclusive
terminology as "solely," "only" and the like in connection with the
recitation of claim elements, or use of a "negative"
limitation.
[0030] It is appreciated that certain features of the invention,
which are, for clarity, described in the context of separate
embodiments, may also be provided in combination in a single
embodiment. Conversely, various features of the invention, which
are, for brevity, described in the context of a single embodiment,
may also be provided separately or in any suitable sub-combination.
All combinations of the embodiments pertaining to the invention are
specifically embraced by the present invention and are disclosed
herein just as if each and every combination was individually and
explicitly disclosed. In addition, all sub-combinations of the
various embodiments and elements thereof are also specifically
embraced by the present invention and are disclosed herein just as
if each and every such sub-combination was individually and
explicitly disclosed herein.
[0031] The publications discussed herein are provided solely for
their disclosure prior to the filing date of the present
application. Nothing herein is to be construed as an admission that
the present invention is not entitled to antedate such publication
by virtue of prior invention. Further, the dates of publication
provided may be different from the actual publication dates which
may need to be independently confirmed.
[0032] The term "pure solution" as used herein refers to a solution
that is substantially free of other components, e.g., a solution
that comprises about 95%, up to about 96%, up to about 97%, up to
about 98%, or up to about 99% or greater of a given compound. For
example, a "pure solution" of methanol would describe a solution
that comprises at least 95%, up to about 96%, up to about 97%, up
to about 98%, up to about 99%, or a greater amount of methanol.
[0033] The term "reagent" as used herein broadly refers to a
chemical compound, a solvent, or a chemical compound dissolved in a
solvent to create a solution. For example, the term "reagent" may
be used herein to describe a thiazin and/or an eosin dye, a
solution comprising a thiazin and/or an eosin dye, or a solvent
(e.g., methanol or de-ionized water).
[0034] The terms "thiazin," "thiazin compound," and "thiazin dye,"
as used interchangeably herein, broadly refer to chemical
substances comprising a ring of four carbon atoms, one nitrogen
atom, and one sulfur atom. Exemplary thiazin compounds include, but
are not limited methylene blue, methylene violet, azure A, azure B,
azure C, thionin, as well as oxidation products of these
compounds.
[0035] The terms "eosin," "eosin compound," and "eosin dye," as
used interchangeably herein, broadly refer to chemical substances
comprising brominated fluorescein. Exemplary eosin compounds
include, but are not limited to, eosin Y and eosin B.
[0036] The term "normal cells" as used herein describes cells
having a clinically ordinary or normal morphology, such as those
that would be found in a subject that does not display symptoms of
a particular disease or disorder.
[0037] The term "abnormal cells" as used herein describes cells
having a clinically relevant abnormal morphology, such as those
that would be found in a subject suffering from a disease or
disorder characterized by abnormal cells that display an altered or
deviated morphology.
[0038] The term "Wright-Giemsa stain," as used herein, generally
refers to a solution comprising thiazin and eosin compounds in
various concentrations. Such solutions may be commercially
available in concentrated or diluted form, or may be created by
dissolving a specified amount of thiazin and/or eosin compounds in
a suitable solvent to create such a solution.
[0039] The term "May-Gr nwald stain," as used herein, generally
refers to a solution comprising thiazin and eosin compounds in
various concentrations. Such solutions may be commercially
available in concentrated or diluted form, or may be created by
dissolving a specified amount of thiazin and/or eosin compounds in
a suitable solvent to create such a solution.
Methods of Use
[0040] The methods of the present disclosure generally find use in
staining biological samples for microscopic analysis, such as,
e.g., examination of the morphology of different cell types present
in a biological sample, in a reduced amount of time. Existing
tissue staining techniques generally involve extended periods of
reagent incubation time, and are not designed to expedite the
staining procedure in order to enable high throughput staining of
multiple samples. The present inventors have discovered a method
for staining biological samples with a Wright-Giemsa staining
reagent or a May-Gr nwald staining reagent that greatly reduces the
overall period of time required to complete the stain as compared
to other known methods. The accelerated staining procedures produce
stained samples having similar quality to those produced using
other known methods. Furthermore, the accelerated staining methods
are highly reproducible, lending themselves to implementation via
manual as well as automated formats.
[0041] The steps involved with the subject methods include, but are
not limited to, applying a biological sample to a substrate,
contacting the sample with a fixation reagent, contacting the
sample with a Wright-Giemsa staining reagent or a May-Gr nwald
staining reagent, and contacting the sample with one or more rinse
reagents. The period of time that the sample is contacted with a
particular reagent is tightly controlled so that the staining
procedure can be carried out in as little time as possible while
still maintaining the quality of the stain. In some embodiments,
the sample is dried. The steps of the subject methods are described
in further detail herein.
Application of Sample to Substrate
[0042] A variety of techniques may be used to apply a biological
sample to a substrate for use in the subject methods. In some
embodiments, a biological sample is applied to the substrate using
a drop-wise application procedure, wherein drops of the sample are
placed on a substrate. In some embodiments, a biological sample is
applied to a substrate by spreading the biological sample in a thin
film over the substrate. In some embodiments, following application
of the sample to the substrate, a spreading or smearing technique
is used to create a thin film of the sample over the surface of the
substrate. In some embodiments, the biological sample is dried on
the substrate before further steps of the methods are carried
out.
[0043] In some embodiments, a biological sample is applied to a
substrate manually by a technician. In some embodiments, a
biological sample is applied to a substrate using automated
equipment, such as, e.g., a smearing machine or apparatus. In some
embodiments, a biological sample is applied to a substrate using a
combination of manual application procedures and automated
application procedures, such as, e.g., wherein a technician applies
one or more drops of a sample to a substrate, and automated
equipment is used to spread the sample over the substrate to create
a thin film.
Contacting the Sample with Reagents
[0044] Following application of a biological sample to a substrate,
the sample is contacted with various reagents in order to carry out
the staining procedure and prepare the sample for microscopic
analysis. In some embodiments, contacting the sample with a reagent
generally involves immersing the substrate in a solution comprising
the reagent, e.g., immersing the substrate in a reagent bath. FIG.
1 is an illustration of a cartridge (or slide carrier) that can be
used to hold multiple substrates (e.g., microscope slides) at the
same time. Such a cartridge may be used to immerse multiple
substrates in a reagent bath at the same time. In other
embodiments, contacting the sample with a reagent involves applying
a sufficient quantity of a solution comprising the reagent to the
substrate in order to cover the biological sample on the
substrate.
[0045] When contacting a sample with a reagent, the substrate may
be positioned in any of a variety of different orientations. For
example, the substrate may be positioned horizontally, vertically,
or may be positioned at an angle with respect to the reagent. In
some embodiments, the substrate is positioned horizontally or at an
angle and a solution comprising the reagent is applied from above
the substrate by dripping, spraying, or otherwise applying the
solution over the surface of the substrate. In some embodiments,
the substrate is positioned vertically and contacting the sample
with a reagent comprises immersing the substrate in a solution
comprising the reagent, e.g., immersing the substrate in a reagent
bath.
[0046] In some embodiments, a combination of reagent application
techniques is used to perform the subject methods. For example, in
some embodiments, a first reagent is applied to the sample by
orienting the substrate vertically and immersing the substrate in a
solution comprising the first reagent. Subsequently, the substrate
is oriented horizontally or at an angle and a solution comprising
another reagent is applied to the sample by dripping, spraying, or
otherwise applying the solution over the surface of the substrate.
Any of a variety of techniques for contacting a biological sample
with a reagent can be combined in any suitable fashion to carry out
the subject methods.
[0047] In some embodiments, the substrate on which the sample is
mounted is held still in the reagent bath during the period of time
in which the sample is contacted with the reagent. For example, in
some embodiments, the substrate is immersed in a reagent bath and
both the substrate and the reagent bath remain motionless for the
period of time during which the sample is contacted with the
reagent. In certain embodiments, the substrate on which the sample
is mounted is not centrifuged, rotated, or subjected to any other
movements during the period of time in which the sample is
contacted with a particular reagent.
Contact with Fixation Reagent
[0048] Following application of the biological sample to the
substrate, the sample is contacted with a fixation reagent for a
controlled period of time. In some embodiments, the sample is
contacted with a fixation reagent for a period of time that ranges
from about 15 seconds up to about 60 seconds. In some embodiments,
the sample is contacted with a fixation reagent for a period of
time that ranges from about 15 seconds up to about 45 seconds. In
some embodiments, the sample is contacted with a fixation reagent
for a period of time that ranges from about 20 seconds up to about
40 seconds. In some embodiments, the sample is contacted with a
fixation reagent for a period of time that ranges from about 25
seconds up to about 35 seconds. In some embodiments, the sample is
contacted with a fixation reagent for a period of time that ranges
from about 30 seconds up to about 35 seconds.
[0049] The inventors of the subject methods have discovered that
the overall amount of time required to carry out a Wright-Giemsa or
a May-Gr nwald staining procedure of sufficient quality on a
biological sample can be greatly reduced by contacting the
biological sample with a fixation reagent for a period of time
ranging from about 20 seconds, up to about 25 seconds, up to about
30 seconds, up to about 35 seconds, up to about 40 seconds, up to
about 45 seconds, up to about 50 seconds, up to about 55 seconds,
up to about 60 seconds.
Contact with Wright-Giemsa Staining Reagent
[0050] Following contact with a fixation reagent, a sample may be
contacted with a Wright-Giemsa staining reagent for a controlled
period of time. In some embodiments, the sample is contacted with a
Wright-Giemsa staining reagent for a period of time that ranges
from about 15 seconds up to about 60 seconds. In some embodiments,
the sample is contacted with a Wright-Giemsa staining reagent for a
period of time that ranges from about 15 seconds up to about 45
seconds. In some embodiments, the sample is contacted with a
Wright-Giemsa staining reagent for a period of time that ranges
from about 20 seconds up to about 40 seconds. In some embodiments,
the sample is contacted with a Wright-Giemsa staining reagent for a
period of time that ranges from about 25 seconds up to about 35
seconds. In some embodiments, the sample is contacted with a
Wright-Giemsa staining reagent for a period of time that ranges
from about 30 seconds up to about 35 seconds.
[0051] The inventors of the subject methods have discovered that
the overall amount of time required to carry out a Wright-Giemsa
staining procedure of sufficient quality on a biological sample can
be greatly reduced by contacting the biological sample with a
Wright-Giemsa staining reagent for a period of time ranging from
about 20 seconds, up to about 25 seconds, up to about 30 seconds,
up to about 35 seconds, up to about 40 seconds, up to about 45
seconds, up to about 50 seconds, up to about 55 seconds, up to
about 60 seconds.
Contact with May-Gr nwald Staining Reagent
[0052] Following contact with a fixation reagent, a sample may be
contacted with a May-Gr nwald staining reagent for a controlled
period of time. In some embodiments, the sample is contacted with a
May-Gr nwald staining reagent for a period of time that ranges from
about 165 seconds up to about 195 seconds. In some embodiments, the
sample is contacted with a May-Gr nwald staining reagent for a
period of time that ranges from about 170 seconds up to about 190
seconds. In some embodiments, the sample is contacted with a May-Gr
nwald staining reagent for a period of time that ranges from about
175 seconds up to about 185 seconds. In some embodiments, the
sample is contacted with a May-Gr nwald staining reagent for a
period of time that ranges from about 180 seconds up to about 185
seconds.
[0053] The inventors of the subject methods have discovered that
the overall amount of time required to carry out a May-Gr nwald
staining procedure of sufficient quality on a biological sample can
be greatly reduced by contacting the biological sample with a
May-Gr nwald staining reagent for a period of time ranging from
about 170 seconds, up to about 175 seconds, up to about 180
seconds, up to about 185 seconds, up to about 190 seconds.
Contact with Rinse Reagent
[0054] Following contact with a staining reagent, a sample may be
contacted with one or more rinse reagents for a controlled period
of time. In some embodiments, the sample is contacted with a first
rinse reagent for a period of time that ranges from about 105
seconds up to about 135 seconds. In some embodiments, the sample is
contacted with a first rinse reagent for a period of time that
ranges from about 110 seconds up to about 130 seconds. In some
embodiments, the sample is contacted with a first rinse reagent for
a period of time that ranges from about 115 seconds up to about 125
seconds.
[0055] The inventors of the subject methods have discovered that
the overall amount of time required to carry out a staining
procedure of sufficient quality on a biological sample can be
greatly reduced by contacting the biological sample with a first
rinse reagent for a period of time ranging from about 110 seconds,
up to about 115 seconds, up to about 120 seconds, up to about 125
second, or up to about 130 seconds.
[0056] In some embodiments, the sample is contacted with a first
rinse reagent for a period of time that ranges from about 135
seconds up to about 165 seconds. In some embodiments, the sample is
contacted with a first rinse reagent for a period of time that
ranges from about 140 seconds up to about 160 seconds. In some
embodiments, the sample is contacted with a first rinse reagent for
a period of time that ranges from about 145 seconds up to about 155
seconds.
[0057] The inventors of the subject methods have discovered that
the overall amount of time required to carry out a staining
procedure of sufficient quality on a biological sample can be
greatly reduced by contacting the biological sample with a first
rinse reagent for a period of time ranging from about 110 seconds,
up to about 115 seconds, up to about 120 seconds, up to about 125
second, or up to about 130 seconds.
[0058] In some embodiments, a sample is contacted with a second
rinse reagent for a period of time that ranges from about 5 seconds
up to about 45 seconds. In some embodiments, the sample is
contacted with a second rinse reagent for a period of time that
ranges from about 10 seconds up to about 40 seconds. In some
embodiments, the sample is contacted with a second rinse reagent
for a period of time that ranges from about 15 seconds up to about
35 seconds. In some embodiments, the sample is contacted with a
second rinse reagent for a period of time that ranges from about 20
seconds up to about 30 seconds.
[0059] The inventors of the subject methods have discovered that
the overall amount of time required to carry out a staining
procedure of sufficient quality on a biological sample can be
greatly reduced, in some embodiments, by contacting the biological
sample with a second rinse reagent for a period of time ranging
from about 10 seconds, up to about 15 seconds, up to about 20
seconds, up to about 25 second, up to about 30 seconds, up to about
35 seconds, or up to about 40 seconds.
Drying
[0060] In some embodiments, following contacting the sample with
the above-described reagents, the sample is dried. Samples can be
dried using simple air drying techniques, wherein the substrate is
stored in air for a sufficient period of time to allow the sample
to dry. In some embodiments, a substrate may be stored in a drying
chamber wherein the temperature is elevated in order to facilitate
the drying process. Drying chambers may also employ circulating air
produced by, e.g., one or more fans, in order to facilitate the
drying process.
Manual and/or Automated Performance of Methods
[0061] The subject methods may be performed manually by one or more
technicians, or may be performed automatically, using automated
equipment that is designed and/or programmed to carry out the steps
of the subject methods. In some embodiments, the subject methods
may be performed through a combination of manual performance by
technicians and automated performance by equipment.
[0062] For example, in some embodiments, the subject methods are
entirely performed manually by technicians. In other embodiments,
the subject methods are performed entirely by automated equipment
that has been designed and/or programmed to carry out the steps of
the subject methods in a particular order. In some embodiments,
certain steps of the subject methods are performed by technicians,
while certain other steps of the subject methods are performed by
automated equipment that has been designed and/or programmed to
carry out the designated steps. Any combination of manual and/or
automated performance may be used to carry out the subject
methods.
[0063] FIG. 2 shows an illustration of an automated staining system
comprising multiple reagent baths and multiple cartridges, each
cartridge holding multiple samples. In use, the automated system
moves a cartridge between the reagent baths and immerses the
cartridges in each reagent bath for a specified period of time.
FIG. 3 shows an illustration of a staining system wherein a
cartridge enters the system, moves between several reagent baths,
moves to a drying area, and then moves out of the automated
staining system.
Biological Samples
[0064] The subject methods may generally be performed on any of a
variety of biological samples in order to facilitate microscopic
analysis of a sample. Exemplary biological samples include, but are
not limited to, biological fluids, e.g., blood samples and bone
marrow samples.
Blood Samples
[0065] Blood samples for use with the subject methods may be
obtained from a subject by any suitable method, including but not
limited to withdrawing blood from a subject using a needle and
syringe. In some embodiments, blood samples may be collected from a
subject using a finger-stick technique, wherein the subject's skin
is punctured and a sufficient amount of blood is "milked" or
expressed from the puncture. Blood samples suitable for use in the
subject methods include both venous and arterial blood samples. Any
blood sample collection procedure may be readily adapted for use
with the subject methods.
Bone Marrow Samples
[0066] Bone marrow samples for use with the subject methods may be
obtained by any suitable method, including but not limited to
needle aspiration. Bone marrow aspiration samples may be collected
using, e.g., an aspiration needle that is inserted through the
subject's skin and then advanced through the outer portion of a
bone and into the bone marrow cavity. Once the needle has been
placed inside the bone marrow cavity, a syringe attached to the
needle is used to aspirate a liquid sample comprising bone marrow.
Any bone marrow sample collection procedure may be readily adapted
for use with the subject methods.
Substrates
[0067] The subject methods involve applying a biological sample to
a substrate. Substrates amenable for use with the subject methods
are generally rigid or semi-rigid structures made of transparent
material. Such materials include, but are not limited to plastic,
glass, and/or quartz. In some embodiments, the substrate is a
microscope slide made of any suitable material and having any
suitable geometry.
[0068] In some embodiments, the length of the substrate ranges in
size from about 20 mm, up to about 25 mm, up to about 30 mm, up to
about 35 mm, up to about 40 mm, up to about 45 mm, up to about 50
mm, up to about 55 mm, up to about 60 mm, up to about 65 mm, up to
about 70 mm, up to about 75 mm, up to about 80 mm. In some
embodiments, the width of the substrate ranges in size from about
20 mm, up to about 25 mm, up to about 30 mm, up to about 35 mm, up
to about 40 mm, up to about 45 mm, up to about 50 mm, up to about
55 mm, up to about 60 mm, up to about 65 mm, up to about 70 mm, up
to about 75 mm, up to about 80 mm. In some embodiments, the
thickness of the substrate ranges from about 0.5 mm, up to about
0.8 mm, up to about 1 mm, up to about 1.2 mm, up to about 1.5 mm.
In some embodiments, the substrate is a standard microscope slide
that is approximately 75 mm in length, 25 mm in width, and 1 mm
thick.
[0069] In some embodiments, the substrate comprises one or more
surface coatings or surface treatments, such as poly-L-lysine or
silane treatment. In some embodiments, the substrate comprises a
surface coating or surface treatment that renders at least a
portion of the substrate opaque or semi-opaque, such as a frosting
or a glazing treatment. In some embodiments, the substrate
comprises an area that can be used for labeling, such as, e.g., an
end or corner region upon which a suitable label can be placed. In
some embodiments, a substrate is labeled using labeling equipment,
such as, e.g., equipment that applies a label to a surface of the
substrate, etches and/or marks the substrate to create a label, or
prints or applies ink and/or any other suitable marking material to
a surface of the substrate to create a label.
[0070] In some embodiments, the substrate may be labeled manually,
e.g., wherein a technician manually places a label on the
substrate, or wherein a technician writes on or otherwise marks the
substrate to create a label. In some embodiments, a technician
utilizes labeling equipment to label a substrate. In some
embodiments, a substrate is labeled using automated labeling
equipment. In such embodiments, labeling information may be
provided to the automated labeling equipment by a computer
program.
Reagents
[0071] The subject methods involve contacting a biological sample
with a variety of different reagents, which are described in more
detail below. The specific temperature of the reagents described
below does not impact the subject methods. The subject methods may
be performed using reagents at a temperature ranging from about
2-8.degree. C., up to about 15.degree. C., up to about 20.degree.
C., up to about 25.degree. C., or up to about 30.degree. C. without
impacting the quality of the stain produced.
Fixation Reagents
[0072] The subject methods involve contacting a biological sample
with a fixation reagent. Suitable fixation reagents generally
include organic solvents, such as alcohols or acetone, which remove
lipids and dehydrate cells. Exemplary fixation reagents include
methanol, ethanol, acetone, solutions comprising these reagents,
and mixtures thereof. In some embodiments, a solution of pure
methanol is used as a fixation reagent. In some embodiments, a
solution of pure acetone is used as a fixation reagent. In some
embodiments, a solution of pure ethanol is used as a fixation
reagent. In some embodiments, a solution of 95% ethanol and 5%
glacial acetic acid is used as a fixation reagent. In some
embodiments, a solution of 50% methanol and 50% acetone is used as
a fixation reagent. In some embodiments, a solution of 50% methanol
and 50% ethanol is used as a fixation reagent.
Wright-Giemsa Staining Reagent
[0073] In some embodiments, the subject methods involve contacting
a biological sample with a Wright-Giemsa staining reagent.
Wright-Giemsa staining reagents generally comprise varying amounts
of both Wright's stain and Giemsa stain compounds, which both
comprise thiazin and eosin dyes. The thiazin and eosin dyes in a
Wright-Giemsa staining reagent bind to and color various components
of biological samples so that they can be visualized under
microscopic examination. Thiazin dyes generally include, but are
not limited to, methylene blue, methylene violet, azure A, azure B,
azure C, and thionin. Thiazin dyes also include oxidation products
of these compounds. Eosin dyes generally include, but are not
limited to, eosin Y and eosin B. Wright-Giemsa staining reagents
and staining stock solutions comprising various amounts of thiazin
and eosin compounds are commercially available to the public from a
variety of sources and suppliers. In some embodiments, a
Wright-Giemsa staining stock solution may also comprise
glycerin.
[0074] In some embodiments of the subject methods, a
commercially-available Wright-Giemsa staining stock solution is
used as the Wright-Giemsa staining reagent without any further
dilution or modification. In some embodiments, a Wright-Giemsa
staining stock solution is diluted prior to use in the subject
methods by mixing the staining stock solution with a suitable
solvent. In some embodiments, a Wright-Giemsa staining stock
solution is diluted with an organic solvent, e.g., methanol, to
produce a staining solution of a particular concentration for use
in the subject methods. In some embodiments, a Wright-Giemsa
staining stock solution is diluted with an aqueous solvent, e.g.,
de-ionized water or phosphate buffer, to produce a staining
solution of a particular concentration for use in the subject
methods.
[0075] In some embodiments, a Wright-Giemsa staining reagent is
created by mixing a desired amount of thiazin and eosin compounds
together in a suitable solvent to create a solution. In some
embodiments, a commercially-available Wright-Giemsa powder,
comprising specified amounts of thiazin and eosin compounds, is
mixed with a suitable solvent to create a solution that can be used
as a Wright-Giemsa staining reagent in the subject methods.
[0076] In some embodiments, the concentration of thiazin dye in the
Wright-Giemsa staining solution ranges from less than about 1%, up
to about 5%, up to about 10%, up to about 15%, up to about 20%, up
to about 25%, up to about 30%, up to about 35%, up to about 40%, up
to about 45%, up to about 50%, up to about 55%, up to about 60%, up
to about 65%, up to about 70%, up to about 75%, up to about 80%, up
to about 85%, or up to about 90%. In some embodiments, the
concentration of eosin dye in the Wright-Giemsa staining solution
ranges from less than about 1%, up to about 5%, about 10%, up to
about 15%, up to about 20%, up to about 25%, up to about 30%, up to
about 35%, up to about 40%, up to about 45%, up to about 50%, up to
about 55%, up to about 60%, up to about 65%, up to about 70%, up to
about 75%, up to about 80%, up to about 85%, or up to about
90%.
[0077] As discussed above, Wright-Giemsa staining stock solutions
are commercially available to the public from a variety of
suppliers, including, e.g., Wright-Giemsa Stain Solution, catalog
number 08711, sold under the trade name ACCUSTAIN.RTM. by
Sigma-Aldrich. Wescor staining solutions are available through
Wecor, Inc. and are sold under the name Aerospray Hematology Pro
Stain. These reagents are available in the form of Buffered Rinse
5L (pH 6.8 and pH 7.2), Thiazin 500 mL, Eosin 500 mL and Fixative
500 mL (Aerofix, methanol). Methanol, Wright Giemsa stain, and
phosphate buffer (pH 6.8 and pH 7.2) reagents are also available
through VWR Inc (VWR.com) under the brand name Harleco Hematology
Stains and Reagents, EMD Millipore or EMD Chemicals, Inc.
[0078] In some embodiments, the concentration of glycerin in the
Wright-Giemsa staining stock solution ranges from less than about
1% to up to about 5%, up to about 8%, up to about 10%, up to about
12%, or up to about 15%.
May-Gr nwald Staining Reagent
[0079] In some embodiments, the subject methods involve contacting
a biological sample with a May-Gr nwald staining reagent. May-Gr
nwald staining reagents generally comprise varying amounts of
thiazin and eosin dyes. The thiazin and eosin dyes in a May-Gr
nwald staining reagent bind to and color various components of
biological samples so that they can be visualized under microscopic
examination. Thiazin dyes generally include, but are not limited
to, methylene blue, methylene violet, azure A, azure B, azure C,
and thionin. Thiazin dyes also include oxidation products of these
compounds. Eosin dyes generally include, but are not limited to,
eosin Y and eosin B. May-Gr nwald staining reagents and staining
stock solutions comprising various amounts of thiazin and eosin
compounds are commercially available to the public from a variety
of sources and suppliers. In some embodiments, a May-Gr nwald
staining stock solution may also comprise glycerin.
[0080] In some embodiments of the subject methods, a
commercially-available May-Gr nwald staining stock solution is used
as the May-Gr nwald staining reagent without any further dilution
or modification. In some embodiments, a May-Gr nwald staining stock
solution is diluted prior to use in the subject methods by mixing
the staining stock solution with a suitable solvent. In some
embodiments, a May-Gr nwald staining stock solution is diluted with
an organic solvent, e.g., methanol, to produce a staining solution
of a particular concentration for use in the subject methods. In
some embodiments, a May-Gr nwald staining stock solution is diluted
with an aqueous solvent, e.g., de-ionized water or phosphate
buffer, to produce a staining solution of a particular
concentration for use in the subject methods.
[0081] In some embodiments, a May-Gr nwald staining reagent is
created by mixing a desired amount of thiazin and eosin compounds
together in a suitable solvent to create a solution. In some
embodiments, a commercially-available May-Gr nwald powder,
comprising specified amounts of thiazin and eosin compounds, is
mixed with a suitable solvent to create a solution that can be used
as a May-Gr nwald staining reagent in the subject methods.
[0082] In some embodiments, the concentration of thiazin dye in the
May-Gr nwald staining solution is about 10%, up to about 15%, up to
about 20%, up to about 25%, up to about 30%, up to about 35%, up to
about 40%, up to about 45%, up to about 50%, up to about 55%, up to
about 60%, up to about 65%, up to about 70%, up to about 75%, up to
about 80%, up to about 85%, or up to about 90%. In some
embodiments, the concentration of eosin dye in the May-Gr nwald
staining solution is about 10%, up to about 15%, up to about 20%,
up to about 25%, up to about 30%, up to about 35%, up to about 40%,
up to about 45%, up to about 50%, up to about 55%, up to about 60%,
up to about 65%, up to about 70%, up to about 75%, up to about 80%,
up to about 85%, or up to about 90%.
[0083] As discussed above, May-Gr nwald staining stock solutions
are commercially available to the public from a variety of
suppliers, including, e.g., May-Gr nwald Stain, catalog number
89027, sold by Thermo Scientific.
[0084] In some embodiments, the concentration of glycerin in the
May-Gr nwald staining stock solution ranges from less than about 1%
up to about 5%, up to about 8%, up to about 10%, up to about 12%,
or up to about 15%.
Rinse Reagents
[0085] The subject methods generally involve contacting a
biological sample with one or more suitable rinse reagents. Rinse
reagents suitable for use with the subject methods include, but are
not limited to, de-ionized water, phosphate buffer solutions
comprising varying concentrations of monobasic potassium phosphate
and dibasic sodium phosphate dissolved in a suitable aqueous
solvent, e.g., de-ionized water, and pH adjusted to a target pH
value. Addition of a rinse reagent having a specified pH value to
the sample ionizes the thiazin and eosin compounds in the staining
reagent and helps them to develop various degrees of coloration,
which facilitates microscopic examination of the sample.
[0086] In some embodiments, the concentration of monobasic
potassium phosphate in the phosphate buffer ranges from 0% up to
about 5%, or up to about 45%, up to about 46%, up to about 47%, up
to about 48%, up to about 49%, up to about 50%, up to about 51%,
wherein the % is a w/w %. In some embodiments, the concentration of
dibasic sodium phosphate in the phosphate buffer ranges from 0% up
to about 5%, or up to about 49%, up to about 50%, up to about 51%,
up to about 52%, up to about 53%, up to about 54%, up to about 55%,
wherein the % is a w/w %.
[0087] In some embodiments, the pH of the phosphate buffer ranges
from about 5.0, up to about 5.2, up to about 5.4, up to about 5.6,
up to about 5.8, up to about 6.0, up to about 6.1, up to about 6.2,
up to about 6.3, up to about 6.4, up to about 6.5, up to about 6.6,
up to about 6.7, up to about 6.8, up to about 6.9, up to about 7.0,
up to about 7.1, up to about 7.2, up to about 7.3, up to about 7.4,
up to about 7.5, up to about 7.6, up to about 7.8, up to about 7.9,
up to about 8.0, up to about 8.2, up to about 8.4, up to about 8.6,
up to about 8.8, up to about 9.0 pH units.
[0088] In some embodiments, commercially-available phosphate buffer
is used as a rinse reagent. Phosphate buffer is commercially
available to the public from a variety of suppliers, including,
e.g., Wright Stain Phosphate Buffer pH 6.8, catalog number 24989,
from Polysciences, Inc. Wescor staining solutions are available
through Wescor, Inc. and are sold under Aerospray Hematology Pro
Stain. The phosphate buffers are available in the form of Buffered
Rinse 5L (pH 6.8 and pH 7.2).
[0089] In some embodiments, the subject methods involve the use of
a high salt phosphate buffer as a rinse reagent. By "high salt
phosphate buffer" is meant a phosphate buffer solution comprising
from about 5.0 to about 5.5 grams of dibasic sodium phosphate and
from about 5.0 to about 5.5 grams of monobasic potassium phosphate
per liter of solvent.
[0090] An example formulation of a high salt phosphate buffer that
can be used in some embodiments of the present methods is provided
in Table 1, below:
TABLE-US-00001 TABLE 1 Example of high salt phosphate buffer
formulation Units of Quantity Component Name Measurement (per
Liter) High Salt Phosphate Buffer Water for in vitro Mfg. LT 1
Sodium Phosphate Dibasic g 5.338 Potassium Phosphate Monobasic g
5.193 Proclin 300 g 0.6 Proclin 950 g 0.8 Characteristics pH 6.8
.+-. 0.20 Osmolarity 10 .+-. 3 mOsm
[0091] In some embodiments, the subject methods involve the use of
a low salt phosphate buffer as a rinse reagent. By "low salt
phosphate buffer" is meant a phosphate buffer solution comprising
from about 0.1 to about 0.5 grams of dibasic sodium phosphate and
from about 0.1 to about 0.5 grams of monobasic potassium phosphate
per liter of solvent.
[0092] An example formulation of a low salt phosphate buffer that
can be used in some embodiments of the present methods is provided
in Table 2, below:
TABLE-US-00002 TABLE 2 Example of low salt phosphate buffer
formulation Units of Quantity Component Name Measurement (per
Liter) Low Salt Phosphate Buffer Water for in vitro Mfg. LT 1
Sodium Phosphate Dibasic g 0.235 Potassium Phosphate Monobasic g
0.229 Proclin 300 g 0.6 Proclin 950 g 0.8 Characteristics pH 6.8
.+-. 0.20 Osmolarity 10 .+-. 3 mOsm
[0093] Both low salt and high salt phosphate buffer solutions may,
in some embodiments, also contain various additional components,
such as, e.g., antimicrobial agents such as, e.g., Proclin 300,
Proclin 950, and the like in suitable quantities, such as, e.g.,
approximately 0.5 up to about 0.6, up to about 0.7, up to about
0.8, up to about 0.9, up to about 1.0 gram per liter of
solvent.
[0094] Additional examples of rinse reagents include, but are not
limited to, de-ionized water, phosphate buffered saline solution,
and the like. FIG. 4, panel A shows a microscopic image of a sample
that was stained and then rinsed with a high salt phosphate buffer,
and panel B shows a microscopic image of a sample that was stained
and then rinsed with a low salt phosphate buffer. As can be seen,
reducing the salt concentration in the phosphate buffer reduced the
amount of stain precipitation in this embodiment.
[0095] FIG. 5 shows a graph of the stain quality as a function of
the pH value of the rinse solution used to perform a May-Gr nwald
stain. As can be seen from the graph, the stain quality is
relatively constant over a pH range of 5.0 up to 9.0 for the rinse
reagent.
[0096] FIG. 6 shows a graph of the stain quality as a function of
the pH value of the rinse solution used to perform a Wright-Giemsa
stain. As can be seen from the graph, the stain quality is
relatively constant over a pH range of 5.0 up to 9.0 for the rinse
reagent.
Additional Embodiments
[0097] In some embodiments, the present disclosure provides an
automated staining system for performing a stain on one or more
samples, comprising: a sample application subsystem that applies a
sample to a substrate; a fixation reagent bath; a staining reagent
bath; one or more rinse reagent baths; a sample transfer subsystem
for moving the substrate from the sample application subsystem, to
the fixation reagent bath, to the staining reagent bath, and then
to the one or more rinse reagent baths; and a computer readable
storage medium (e.g., a digital storage medium) comprising
instructions executable by at least one processing device that,
when executed, cause the processing device to control the sample
transfer subsystem such that the sample transfer subsystem places
the one or more samples into the reagent baths for a controlled
period of time.
[0098] In some embodiments, the sample is placed in the fixation
reagent bath for a period of time ranging from about 15 seconds, up
to about 20 seconds, up to about 25 seconds, up to about 30
seconds, up to about 35 seconds, up to about 40 seconds, up to
about 45 seconds, up to about 50 seconds, up to about 55 seconds,
up to about 60 seconds. In some embodiments, the fixation reagent
is methanol.
[0099] In some embodiments, the sample is placed in a Wright-Giemsa
staining reagent bath for a period of time ranging from about 15
seconds, up to about 20 seconds, up to about 25 seconds, up to
about 30 seconds, up to about 35 seconds, up to about 40 seconds,
up to about 45 seconds, up to about 50 seconds, up to about 55
seconds, up to about 60 seconds. In some embodiments, the
Wright-Giemsa staining reagent comprises methanol.
[0100] In some embodiments, the sample is placed in a May-Gr nwald
staining reagent bath for a period of time ranging from about 165
seconds, up to about 170 seconds, up to about 175 seconds, up to
about 180 seconds, up to about 185 seconds, up to about 190
seconds, or up to about 195 seconds. In some embodiments, the
May-Gr nwald staining reagent comprises methanol.
[0101] In some embodiments, the sample is placed in a first rinse
reagent bath for a period of time ranging from about 105 seconds,
up to about 110 seconds, up to about 115 seconds, up to about 120
seconds, up to about 125 seconds, up to about 130 seconds, up to
about 135 seconds, up to about 140 seconds, up to about 145
seconds, up to about 150 seconds, up to about 155 seconds, up to
about 160 seconds, up to about 165 seconds. In some embodiments,
the first rinse reagent comprises de-ionized water. In some
embodiments, the first rinse reagent is a phosphate buffer. In some
embodiments, the first rinse reagent is a low salt phosphate buffer
or a high salt phosphate buffer.
[0102] In some embodiments, the phosphate buffer has a pH value
ranging from about 5.0 up to about 9.0 pH units. In some
embodiments, the phosphate buffer has a pH value ranging from about
6.5 up to about 7.2 pH units.
[0103] In some embodiments, a sample is placed in a second rinse
reagent bath for a period of time ranging from about 5 seconds, up
to about 10 seconds, up to about 15 seconds, up to about 20
seconds, up to about 25 seconds, up to about 30 seconds, up to
about 35 seconds, up to about 40 seconds, up to about 45 seconds.
In some embodiments, the second rinse reagent comprises de-ionized
water. In some embodiments, the second rinse reagent is a phosphate
buffer. In some embodiments, the second rinse reagent is a low salt
phosphate buffer or a high salt phosphate buffer.
[0104] In some embodiments, the phosphate buffer has a pH value
ranging from about 5.0 up to about 9.0 pH units. In some
embodiments, the phosphate buffer has a pH value ranging from about
6.5 up to about 7.2 pH units.
[0105] In some embodiments, the sample is placed in a drying
chamber. In some embodiments, the substrate is a glass microscope
slide. In some embodiments, the sample is a biological fluid. In
some embodiments, the sample is a blood sample. In some
embodiments, the sample is a bone marrow sample.
EXAMPLES
[0106] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to make and use the present invention, and are
not intended to limit the scope of what the inventors regard as
their invention nor are they intended to represent that the
experiments below are all or the only experiments performed.
Efforts have been made to ensure accuracy with respect to numbers
used (e.g., amounts, temperature, etc.) but some experimental
errors and deviations should be accounted for. Unless indicated
otherwise, parts are parts by weight, molecular weight is weight
average molecular weight, temperature is in degrees Celsius, and
pressure is at or near atmospheric. Standard abbreviations may be
used, e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s
or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino
acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s);
i.m., intramuscular(ly); i.p., intraperitoneal(ly); s.c.,
subcutaneous(ly); and the like.
Materials and Methods
[0107] The following materials and methods were used to carry out
the examples below.
Stain Quality Scoring Index
[0108] In order to provide an objective assessment of the quality
of a stain produced from a given staining procedure, the present
inventors created a Stain Quality Scoring Index. The numbers in the
scoring index range from 1 to 4, wherein 1 is unacceptable quality,
2 is borderline quality, 3 is acceptable quality, and 4 is optimal
quality. "Unacceptable" describes a stain wherein the observer
cannot differentiate white blood cell features (such as cytoplasmic
granules) and cannot perform an accurate manual differential count.
"Borderline" describes a stain wherein the observer can
differentiate most white blood cell features but may or may not be
able to perform an accurate manual differential count. "Acceptable"
describes a stain wherein all white blood cells are appropriately
stained and the observer can perform an accurate manual
differential count on both normal and abnormal cells. "Optimal"
describes a stain having ideal stain quality that allows for an
easy and accurate manual differential count on both normal and
abnormal cell types.
[0109] Stained samples were microscopically examined by medical
technicians and pathologists and were scored in a variety of
general categories, such as staining consistency across the
monolayer and stain integrity (inclusion of precipitates, dust,
staining artifacts, cells washed away or damaged, etc.), as well as
a variety of specific categories, including, e.g., an assessment of
the stain quality on neutrophils, lymphocytes, monocytes,
eosinophils, basophils, erythrocytes, promyelocytes, myelocytes,
metamyelocytes, dohle bodies, howell-jolly bodies, large platelets,
and the like present in the sample. Assigned numerical scores were
then used to compare the quality of stains produced by different
staining procedures and to evaluate the reproducibility of staining
procedures.
Example 1
Acceptability of Accelerated Wright-Giemsa Staining Procedure with
Normal Cells
[0110] An accelerated Wright-Giemsa staining procedure was
developed wherein a sample was applied to a substrate and contacted
with methanol as a fixation reagent for 30 seconds, followed by
contact with a Wright-Giemsa staining reagent for 30 seconds,
followed by contact with phosphate buffer at pH 6.8 for 120
seconds, followed by contact with de-ionized water for 15 seconds,
followed by air drying. A sample containing normal cells (i.e.,
cells with clinically normal morphology) was subjected to this
staining procedure and evaluated by 14 different individuals
experienced in examining hematology samples. These individuals used
the Stain Quality Scoring Index described above to assign numerical
scores to the sample in a variety of categories. The numerical
scores in each category were then used to create an overall average
numerical score for the sample.
[0111] In parallel, the same individuals also performed a standard
hematology stain on the same sample using their own preferred
procedure. The standard staining procedures generally involved
longer reagent exposure times for each of the reagents as compared
to the accelerated staining procedure described above. The samples
that were stained using the standard staining procedure were also
evaluated using the Stain Quality Scoring Index and assigned an
overall average numerical score.
[0112] The results from the analysis of the two samples that
underwent different staining procedures were then compared for each
of the evaluating individuals. The data are provided in Table 3,
below.
TABLE-US-00003 TABLE 3 Comparison of numerical score assigned to
normal cells stained with standard staining procedure and
accelerated Wright-Giemsa staining procedure. Individual 1 2 3 4 5
6 7 8 9 10 11 12 13 14 Standard Staining 3.8 3.3 3.9 n/a 3.9 3.3
3.8 2.0 3.9 3.6 4.0 2.7 2.6 3.7 Procedure Accelerated 3.8 4.0 4.0
3.8 3.3 n/a 3.8 3.0 4.0 3.8 4.0 3.9 3.4 3.8 Wright-Giemsa Staining
Procedure
Example 2
Acceptability of Accelerated Wright-Giemsa Staining Procedure with
Abnormal Cells
[0113] An accelerated Wright-Giemsa staining procedure was
developed wherein a sample was applied to a substrate and contacted
with methanol as a fixation reagent for 30 seconds, followed by
contact with a Wright-Giemsa staining reagent for 30 seconds,
followed by contact with phosphate buffer at pH 6.8 for 120
seconds, followed by contact with de-ionized water for 15 seconds,
followed by air drying. A sample containing abnormal cells (i.e.,
cells with clinically abnormal morphology) was subjected to this
staining procedure and evaluated by 14 different individuals
experienced in examining hematology samples. These individuals used
the Stain Quality Scoring Index described above to assign numerical
scores to the sample in a variety of categories. The numerical
scores in each category were then used to create an overall average
numerical score for the sample.
[0114] In parallel, the same individuals also performed a standard
hematology stain on the same sample using their own preferred
procedure. The standard staining procedures generally involved
longer reagent exposure times for each of the reagents as compared
to the accelerated staining procedure described above. The samples
that were stained using the standard staining procedure were also
evaluated using the Stain Quality Scoring Index and assigned an
overall average numerical score.
[0115] The results from the analysis of the two samples that
underwent different staining procedures were then compared for each
of the evaluating individuals. The data are provided in Table 4,
below.
TABLE-US-00004 TABLE 4 Comparison of numerical score assigned to
abnormal cells stained with standard staining procedure and
accelerated Wright-Giemsa staining procedure. Individual 1 2 3 4 5
6 7 8 9 10 11 12 13 14 Standard Staining 2.0 3.3 3.4 n/a 2.1 3.1
2.9 2.0 2.8 3.6 3.8 2.4 2.9 3.0 Procedure Accelerated 3.6 4.0 4.0
4.0 2.8 n/a 2.3 3.8 4.0 3.7 3.6 2.9 1.5 3.4 Wright-Giemsa Staining
Procedure
Example 3
Reproducibility of Accelerated Wright-Giemsa and May-Gr nwald
Staining Procedure with Normal Cells
[0116] The reproducibility of the accelerated Wright-Giemsa
staining procedure described above in Examples 1 and 2 was
evaluated by repeatedly performing the staining procedure on 15
separate days. A sample of normal cells was stained in triplicate
on each day using the accelerated procedure, and the samples were
assessed for the staining quality on erythrocytes, eosinophilic
granules, basophilic granules, leukocyte nuclei, leukocyte
cytoplasm, nutrophilic granules, and platelets using the Stain
Quality Scoring Index. The results are summarized below in Table
5.
[0117] The reproducibility of the accelerated Wright-Giemsa
staining procedure was also tested by repeated staining a number of
samples over a period of days and plotting the observed stain
quality score on each day. The results are summarized in FIG. 7,
panel A.
[0118] The reproducibility of the accelerated May-Gr nwald staining
procedure was also tested by repeated staining a number of samples
over a period of days and plotting the observed stain quality score
on each day. The results are summarized in FIG. 7, panel B.
[0119] FIG. 8, panel A shows the individual cell ratings for
samples that were stained using an accelerated Wright-Giemsa
staining procedure of the present disclosure. Panel B shows the
individual cell ratings for samples that were stained using an
accelerated May-Gr nwald staining procedure of the present
disclosure. The results indicate that the staining procedures
produce highly reproducible staining quality for the cells types
that were examined.
TABLE-US-00005 TABLE 5 Repeatability data from normal samples
stained using accelerated Wright- Giemsa staining procedure.
Repeatability Results Day 1 Day 1 Day 1 Day 2 Day 2 Day 2 Day 3 Day
3 Day 3 Normal Cells Slide 1 Slide 2 Slide 3 Slide 1 Slide 2 Slide
3 Slide 1 Slide 2 Slide 3 Erythocytes 4 4 4 4 4 4 3 4 4
Eosinophilic Granules 4 4 N/A 4 4 4 1 4 4 Basophilic Granules 4 4 4
N/A N/A 4 N/A N/A 4 Leukocyte Nuclei 4 4 4 4 4 4 3 4 4 Leukocyte
Cytoplasm 4 4 4 4 4 4 1 4 4 Nutrophilic Granules 4 4 4 4 4 4 1 4 4
Platelets 4 4 4 4 4 4 3 4 4 Day 4 Day 4 Day 4 Day 5 Day 5 Day 5 Day
6 Day 6 Day 6 Slide 1 Slide 2 Slide 3 Slide 1 Slide 2 Slide 3 Slide
1 Slide 2 Slide 3 Erythocytes 4 4 4 4 4 4 4 4 4 Eosinophilic
Granules 4 4 4 4 4 4 4 4 4 Basophilic Granules N/A N/A N/A 4 4 4 4
4 4 Leukocyte Nuclei 4 4 4 4 4 4 4 4 4 Leukocyte Cytoplasm 4 4 4 4
4 4 4 4 4 Nutrophilic Granules 4 4 4 4 4 4 4 4 4 Platelets 4 4 4 4
4 4 4 4 4 Day 7 Day 7 Day 7 Day 8 Day 8 Day 8 Day 9 Day 9 Day 9
Slide 1 Slide 2 Slide 3 Slide 1 Slide 2 Slide 3 Slide 1 Slide 2
Slide 3 Erythocytes 4 4 4 4 4 4 4 4 4 Eosinophilic Granules 4 4 4 4
4 4 4 4 4 Basophilic Granules 4 4 4 N/A N/A N/A N/A N/A 4 Leukocyte
Nuclei 4 4 4 4 4 4 4 4 4 Leukocyte Cytoplasm 4 4 4 4 4 4 4 4 4
Nutrophilic Granules 4 4 4 4 4 4 4 4 4 Platelets 4 4 4 4 4 4 4 4 4
Day 10 Day 10 Day 10 Day 11 Day 11 Day 11 Day 12 Day 12 Day 12
Slide 1 Slide 2 Slide 3 Slide 1 Slide 2 Slide 3 Slide 1 Slide 2
Slide 3 Erythocytes 4 4 4 4 4 4 4 4 4 Eosinophilic Granules 4 4 4 4
4 4 4 4 4 Basophilic Granules 4 4 4 N/A N/A N/A N/A 4 4 Leukocyte
Nuclei 4 4 4 4 4 4 4 4 4 Leukocyte Cytoplasm 4 4 4 4 4 4 4 4 4
Nutrophilic Granules 4 4 4 4 4 4 4 4 4 Platelets 4 4 4 4 4 4 4 4 4
Day 13 Day 13 Day 13 Day 14 Day 14 Day 14 Day 15 Day 15 Day 15
Slide 1 Slide 2 Slide 3 Slide 1 Slide 2 Slide 3 Slide 1 Slide 2
Slide 3 Erythocytes 4 4 4 4 4 4 4 4 4 Eosinophilic Granules 4 4 4 4
4 4 4 4 4 Basophilic Granules 4 N/A N/A 4 4 4 N/A N/A N/A Leukocyte
Nuclei 4 4 4 4 4 4 4 4 4 Leukocyte Cytoplasm 4 4 4 4 4 4 4 4 4
Nutrophilic Granules 4 4 4 4 4 4 4 4 4 Platelets 4 4 4 4 4 4 4 4
4
Example 4
Reproducibility of Accelerated Wright-Giemsa Staining Procedure
with Abnormal Cells
[0120] The reproducibility of the accelerated Wright-Giemsa
staining procedure described above in Examples 1 and 2 was
evaluated by repeatedly performing the staining procedure on 15
separate days. A sample of abnormal cells was stained in triplicate
on each day using the accelerated procedure, and the samples were
assessed for the staining quality on erythrocytes, eosinophilic
granules, basophilic granules, leukocyte nuclei, leukocyte
cytoplasm, nutrophilic granules, and platelets using the Stain
Quality Scoring Index. The results are summarized below in Table
6.
TABLE-US-00006 TABLE 6 Repeatability data from abnormal samples
stained using accelerated Wright- Giemsa staining procedure.
Repeatability Results Day 1 Day 1 Day 1 Day 2 Day 2 Day 2 Day 3 Day
3 Day 3 Abnormal Cells Slide 1 Slide 2 Slide 3 Slide 1 Slide 2
Slide 3 Slide 1 Slide 2 Slide 3 Erythocytes 4 4 4 4 4 4 4 4 4
Eosinophilic Granules N/A N/A N/A N/A N/A N/A N/A N/A N/A
Basophilic Granules 4 4 4 N/A N/A N/A N/A N/A N/A Leukocyte Nuclei
4 4 4 4 4 4 4 4 4 Leukocyte Cytoplasm 4 4 4 4 4 4 4 4 4 Nutrophilic
Granules 4 4 4 4 4 4 4 4 4 Platelets 4 4 4 4 4 4 4 4 4 Day 4 Day 4
Day 4 Day 5 Day 5 Day 5 Day 6 Day 6 Day 6 Slide 1 Slide 2 Slide 3
Slide 1 Slide 2 Slide 3 Slide 1 Slide 2 Slide 3 Erythocytes 4 4 4 4
4 4 4 4 4 Eosinophilic Granules N/A 4 N/A N/A N/A N/A N/A N/A N/A
Basophilic Granules N/A N/A 4 N/A N/A N/A N/A N/A N/A Leukocyte
Nuclei 4 4 4 4 4 4 4 2 4 Leukocyte Cytoplasm 4 4 4 4 4 4 4 2 4
Nutrophilic Granules 4 4 4 4 4 4 4 2 4 Platelets 4 4 4 4 4 4 4 4 4
Day 7 Day 7 Day 7 Day 8 Day 8 Day 8 Day 9 Day 9 Day 9 Slide 1 Slide
2 Slide 3 Slide 1 Slide 2 Slide 3 Slide 1 Slide 2 Slide 3
Erythocytes 4 4 4 4 4 4 4 4 4 Eosinophilic Granules 4 4 4 4 4 4 4 4
4 Basophilic Granules 4 4 4 4 4 N/A 4 N/A 4 Leukocyte Nuclei 4 4 4
4 4 4 4 4 4 Leukocyte Cytoplasm 4 4 4 4 4 4 4 4 4 Nutrophilic
Granules 4 4 4 4 4 4 4 4 4 Platelets 4 4 4 4 4 4 4 4 4 Day 10 Day
10 Day 10 Day 11 Day 11 Day 11 Day 12 Day 12 Day 12 Slide 1 Slide 2
Slide 3 Slide 1 Slide 2 Slide 3 Slide 1 Slide 2 Slide 3 Erythocytes
4 4 4 4 4 4 4 4 4 Eosinophilic Granules N/A N/A N/A N/A N/A N/A N/A
N/A N/A Basophilic Granules N/A N/A N/A N/A N/A N/A N/A N/A N/A
Leukocyte Nuclei 4 4 4 4 4 4 4 4 4 Leukocyte Cytoplasm 4 4 4 4 4 4
4 4 4 Nutrophilic Granules 4 4 4 4 4 4 4 4 4 Platelets 4 4 4 4 4 4
4 4 4 Day 13 Day 13 Day 13 Day 14 Day 14 Day 14 Day 15 Day 15 Day
15 Slide 1 Slide 2 Slide 3 Slide 1 Slide 2 Slide 3 Slide 1 Slide 2
Slide 3 Erythocytes 4 4 3 4 4 4 4 4 4 Eosinophilic Granules N/A N/A
N/A 4 4 4 4 4 4 Basophilic Granules N/A N/A N/A 4 4 N/A N/A N/A N/A
Leukocyte Nuclei 4 4 3 4 4 4 4 4 4 Leukocyte Cytoplasm 4 4 3 4 4 4
4 4 4 Nutrophilic Granules 4 4 3 4 4 4 4 4 4 Platelets 4 4 3 4 4 4
4 4 4
Example 5
Acceptability of Accelerated May-Gr nwald Staining Procedure with
Normal Cells
[0121] An accelerated May-Gr nwald staining procedure was developed
wherein a sample was applied to a substrate and contacted with
methanol as a fixation reagent for 30 seconds, followed by contact
with a May-Gr nwald staining reagent for 180 seconds, followed by
contact with phosphate buffer at pH 6.8 for 150 seconds, followed
by contact with de-ionized water for 30 seconds, followed by air
drying. A sample containing normal cells (i.e., cells with
clinically normal morphology) was subjected to this staining
procedure and evaluated by 14 different individuals experienced in
examining hematology samples. These individuals used the Stain
Quality Scoring Index described above to assign numerical scores to
the sample in a variety of categories. The numerical scores in each
category were then used to create an overall average numerical
score for the sample.
[0122] In parallel, the same individuals also performed a standard
hematology stain on the same sample using their own preferred
procedure. The standard staining procedures generally involved
longer reagent exposure times for each of the reagents as compared
to the accelerated staining procedure described above. The samples
that were stained using the standard staining procedure were also
evaluated using the Stain Quality Scoring Index and assigned an
overall average numerical score.
[0123] The results from the analysis of the two samples that
underwent different staining procedures were then compared for each
of the evaluating individuals. The data are provided in Table 7,
below.
TABLE-US-00007 TABLE 7 Comparison of numerical score assigned to
normal cells stained with standard staining procedure and
accelerated May-Grunwald staining procedure. Individual 1 2 3 4 5 6
7 8 9 10 11 12 13 14 Standard Staining 3.8 3.3 3.9 n/a 3.9 3.3 3.8
2.0 3.9 3.6 4.0 2.7 2.6 3.7 Procedure Accelerated May- 3.1 3.4 3.4
3.9 3.5 3.9 3.1 2.6 4.0 3.2 3.7 3.8 3.0 3.7 Grunwald Staining
Procedure
Example 6
Acceptability of Accelerated May-Gr nwald Staining Procedure with
Abnormal Cells
[0124] An accelerated May-Gr nwald staining procedure was developed
wherein a sample was applied to a substrate and contacted with
methanol as a fixation reagent for 30 seconds, followed by contact
with a May-Gr nwald staining reagent for 180 seconds, followed by
contact with phosphate buffer at pH 6.8 for 150 seconds, followed
by contact with de-ionized water for 30 seconds, followed by air
drying. A sample containing abnormal cells (i.e., cells with
clinically abnormal morphology) was subjected to this staining
procedure and evaluated by 14 different individuals experienced in
examining hematology samples. These individuals used the Stain
Quality Scoring Index described above to assign numerical scores to
the sample in a variety of categories. The numerical scores in each
category were then used to create an overall average numerical
score for the sample.
[0125] In parallel, the same individuals also performed a standard
hematology stain on the same sample using their own preferred
procedure. The standard staining procedures generally involved
longer reagent exposure times for each of the reagents as compared
to the accelerated staining procedure described above. The samples
that were stained using the standard staining procedure were also
evaluated using the Stain Quality Scoring Index and assigned an
overall average numerical score.
[0126] The results from the analysis of the two samples that
underwent different staining procedures were then compared for each
of the evaluating individuals. The data are provided in Table 8,
below.
TABLE-US-00008 TABLE 8 Comparison of numerical score assigned to
abnormal cells stained with standard staining procedure and
accelerated May-Grunwald staining procedure. Individual 1 2 3 4 5 6
7 8 9 10 11 12 13 14 Standard May- 2.0 3.3 3.4 n/a 2.1 3.1 2.9 2.0
2.8 3.6 3.8 2.4 2.9 3.0 Grunwald Staining Procedure Accelerated
May- 2.3 2.2 2.1 3.9 2.4 3.7 2.6 2.6 3.0 2.9 2.3 2.4 2.6 2.3
Grunwald Staining Procedure
Example 7
Accelerated May-Gr nwald Staining Procedure Using De-Ionized Water
and No Phosphate Buffer
[0127] An accelerated May-Gr nwald staining procedure was developed
wherein a sample was applied to a microscope slide, contacted with
methanol as a fixation reagent for 30 seconds and contacted with a
May-Gr nwald staining reagent for 180 seconds. Following contact
with the May-Gr nwald staining reagent, the slide was evenly coated
with de-ionized water without draining the May-Gr nwald staining
reagent, and the slide was then allowed to sit for 90 seconds.
Next, the slide was rinsed for 10 seconds in de-ionized water. The
slide was then dried and examined. Microscopic examination revealed
that the sample has been successfully stained.
INCORPORATION BY REFERENCE
[0128] This application is related to co-pending and co-owned
provisional applications: AUTOMATED SMEAR MAKING APPARATUS (U.S.
Provisional Patent Application Ser. No. 61/581,032) and MICROSCOPE
SLIDE CARRIER (U.S. Provisional Patent Application Ser. No.
61/581,037), the disclosures of which are herein incorporated by
reference in their entirety.
[0129] While the present invention has been described with
reference to the specific embodiments thereof, it should be
understood by those skilled in the art that various changes may be
made and equivalents may be substituted without departing from the
true spirit and scope of the invention. In addition, many
modifications may be made to adapt a particular situation,
material, composition of matter, process, process step or steps, to
the objective, spirit and scope of the present invention. All such
modifications are intended to be within the scope of the claims
appended hereto.
* * * * *